Many (20%-40%) cancer patients develop brain metastases, a condition with a typical life expectancy of only 3-6 months. Chemotherapy is seldom effective because systemically administered drugs reach brain metastases in very low concentrations due to (i) the blood-tumor barrier (BTB), which is created by heterogeneous vessel permeability and high interstitial pressure, (ii) the blood-brain barrier (BBB), which protects infiltrating cancer cells on tumor margins, and (iii) the nanoporous and electrostaticaly charged tissue space, which limits diffusion. Until effective strategies for overcoming these barriers emerge, brain metastasis patients will not benefit from immunotherapy. To address this problem, we have assembled a team of engineers, biologists, and clinicians with expertise in image-guided drug delivery, nanomedicine, and immunotherapy. Our goal is to engineer image-guided immunotherapies for brain metastases by combining technologies for targeted BTB/BBB opening (MRI-guided focused ultrasound and microbubbles) and improved brain-tissue penetration (nanoparticles with densely PEGylated bioinert surfaces) with strategies for enhancing T-cell infiltration and cytotoxic anti-tumor function. Our approach is supported by preliminary studies showing, for the first time, robust and sustained transgene expression in the brain via the delivery of non-viral nanoparticles with focused ultrasound. This proposal consists of 3 aims. In Aim 1, we will develop the therapeutic delivery platform, whereby intracerebral B16 melanoma is effectively transfected via the focused ultrasound-mediated delivery of brain- penetrating nanoparticles. Aim 1a will be to engineer nanoparticles that effectively penetrate and transfect B16 tumors, while Aims 1b and 1c will be to develop an MRI-guided approach for safe and effective. In Aim 2, we propose to inhibit immunosuppression and control tumor growth by the focused ultrasound-targeted delivery of signal transducer and activator of transcription 3 (STAT3)-interfering brain-penetrating nanoparticles. STAT3 is constitutively active in melanoma metastases and critical in many immunosuppressive pathways, thus STAT3 RNA interference (RNAi) should be amongst the most highly effective methods for boosting tumor immunity and inhibiting growth. Finally, in Aim 3, we propose to enhance anti-tumor immune cell infiltration and function and control tumor growth by the ultrasound-targeted delivery of brain-penetrating nanoparticles that encode an antigen recognized as foreign. In essence, our objective is to elicit an immunotherapeutic memory recall response against B16 via the high-efficiency transfection of tumor and dendritic cells with an antigen (M1 matrix protein) recognized as foreign by the host (influenza-immunized HLA-A2 mice, which recognize influenza through M1). We postulate that M1 transfection will synergize with enhanced intratumor immune cell infiltration during BBB opening to elicit a memory recall response, including the exponential expansion of potent M1-specific secondary effector T-cell populations within the tumor and deep cervical lymph nodes.